George Kemble

Inhibition of de novo Palmitate Synthesis by Fatty Acid Synthase Induces Apoptosis in Tumor Cells by Remodeling Cell Membranes, Inhibiting Signaling Pathways, and Reprogramming Gene Expression

Inhibition of de novo palmitate synthesis via fatty acid synthase (FASN) inhibition provides an unproven approach to cancer therapy with a strong biological rationale. FASN expression increases with tumor progression and associates with chemoresistance, tumor metastasis, and diminished patient survival in numerous tumor types. TVB-3166, an orally-available, reversible, potent, and selective FASN inhibitor induces apoptosis, inhibits anchorage-independent cell growth under lipid-rich conditions, and inhibits in-vivo xenograft tumor growth. Dose-dependent effects are observed between 20–200 nM TVB-3166, which agrees with the IC50 in biochemical FASN and cellular palmitate synthesis assays. Mechanistic studies show that FASN inhibition disrupts lipid raft architecture, inhibits biological pathways such as lipid biosynthesis, PI3K–AKT–mTOR and β-catenin signal transduction, and inhibits expression of oncogenic effectors such as c-Myc; effects that are tumor-cell specific. Our results demonstrate that FASN inhibition has anti-tumor activities in biologically diverse preclinical tumor models and provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers, including those expressing mutant K-Ras, ErbB2, c-Met, and PTEN. The reported findings inform ongoing studies to link mechanisms of action with defined tumor types and advance the discovery of biomarkers supporting development of FASN inhibitors as cancer therapeutics. Research in context Fatty acid synthase (FASN) is a vital enzyme in tumor cell biology; the over-expression of FASN is associated with diminished patient prognosis and resistance to many cancer therapies. Our data demonstrate that selective and potent FASN inhibition with TVB-3166 leads to selective death of tumor cells, without significant effect on normal cells, and inhibits in vivo xenograft tumor growth at well-tolerated doses. Candidate biomarkers for selecting tumors highly sensitive to FASN inhibition are identified. These preclinical data provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers.

Diacylglycerol Metabolism and Signaling Is a Driving Force Underlying FASN Inhibitor Sensitivity in Cancer Cells

Fatty acid synthase (FASN) generates the de novo source of lipids for cell proliferation and is a promising cancer therapy target. Development of FASN inhibitors, however, necessitates a better understanding of sensitive and resistant cancer types to optimize patient treatment. Indeed, testing the cytotoxic effects of FASN inhibition across human cancer cells revealed diverse sensitivities. We show here that metabolic incorporation of glucose into specific complex lipid species strongly predicts FASN inhibitor sensitivity. We also show that the levels of one of these lipid classes, protein kinase C (PKC) stimulator diacylglycerols, are lowered upon FASN inhibitor treatment in sensitive compared to resistant cells and that PKC activators and inhibitors rescue cell death in sensitive cells and sensitize resistant cells, respectively. Our findings not only reveal a biomarker for predicting FASN sensitivity in cancer cells but also a put forth a heretofore unrecognized mechanism underlying the anticancer effects of FASN inhibitors.

FASN Inhibition and Taxane Treatment Combine to Enhance Anti-tumor Efficacy in Diverse Xenograft Tumor Models through Disruption of Tubulin Palmitoylation and Microtubule Organization and FASN Inhibition-Mediated Effects on Oncogenic Signaling and Gene Expression

Palmitate, the enzymatic product of FASN, and palmitate-derived lipids support cell metabolism, membrane architecture, protein localization, and intracellular signaling. Tubulins are among many proteins that are modified post-translationally by acylation with palmitate. We show that FASN inhibition with TVB-3166 or TVB-3664 significantly reduces tubulin palmitoylation and mRNA expression. Disrupted microtubule organization in tumor cells is an additional consequence of FASN inhibition. FASN inhibition combined with taxane treatment enhances inhibition of in vitro tumor cell growth compared to treatment with either agent alone. In lung, ovarian, prostate, and pancreatic tumor xenograft studies, FASN inhibition and paclitaxel or docetaxel combine to inhibit xenograft tumor growth with significantly enhanced anti-tumor activity. Tumor regression was observed in 3 of 6 tumor xenograft models. FASN inhibition does not affect cellular taxane concentration in vitro. Our data suggest a mechanism of enhanced anti-tumor activity of the FASN and taxane drug combination that includes inhibition of tubulin palmitoylation and disruption of microtubule organization in tumor cells, as well as a sensitization of tumor cells to FASN inhibition-mediated effects that include gene expression changes and inhibition of β-catenin. Together, the results strongly support investigation of combined FASN inhibition and taxane treatment as a therapy for a variety of human cancers.

Abstract LB-214: FASN inhibitor TVB-2640 shows pharmacodynamic effect and evidence of clinical activity in KRAS-mutant NSCLC patients in a phase I study

TVB-2640 is an oral, first-in-class, selective and reversible inhibitor of fatty acid synthase (FASN) in Phase 1 testing in solid tumor patients (study 3V2640-CLIN-002). FASN is a central mediator of neoplastic lipogenesis and uniquely catalyzes the production of palmitate, a key signaling molecule and the building block for long chain fatty acids. Tumor cells frequently increase FASN expression compared to normal cells to support an increased dependence on de novo lipogenesis to produce palmitate, phospholipids, lipid second messengers, membranes and lipid rafts for oncogenic signaling. High FASN correlates with poor prognosis in several tumor types including NSCLC. Palmitoylation, the reversible attachment of palmitate to proteins regulates membrane interactions and dynamically alters protein trafficking and function. RAS proteins require palmitoylation for plasma membrane localization and signaling, and KRAS4A mutated at G12 specifically requires palmitoylation for oncogenic activity. FASN inhibitors may therefore provide a novel approach to target KRAS which to date has eluded many drug development approaches. In vitro viability assays showed that KRAS mutant NSCLC cell lines show greater sensitivity to FASN inhibitors than KRAS WT. Furthermore gene expression analysis of TCGA NSCLC tumors and PDX models showed that KRAS mutant cases are associated with a higher incidence of lipogenic features than KRAS WT. The ongoing Phase 1 study includes 2 NSCLC expansion cohorts; TVB-2640 as monotherapy or in combination with paclitaxel. Several biomarker approaches are ongoing to investigate pharmacodynamic activity and patient enrichment strategies. Metabolomic profiling of serum from 12 NSCLC patients showed increased serum malonyl carnitine and decreased palmitate- derived lipids after 8 or 15 days of TVB-2640 treatment. These changes are consistent with FASN inhibition, and also observed in preclinical models. A novel non-invasive approach using Sebutape patches to collect forehead sebum showed inhibition of de novo lipogenesis in 7/7 patients including 2 NSCLC. Decreases in wax esters, saturated and monounsaturated triglycerides were observed, indicating that these lipids are not restored by diet. To date, 16 NSCLC patients are considered evaluable for clinical activity; 6 have KRAS mutant tumors, 2 have wild type KRAS and 8 are unknown. Of the 6 known KRAS mutant patients, 3 are in the monotherapy cohort and all 3 have stable disease for 20, >25 and >37 weeks respectively. The remaining 3 KRAS mutant patients are on combination therapy; one achieved a partial response and remains on therapy (>22 weeks), 1 had stable disease (24 weeks), and 1 progressed. This study shows that TVB-2640 1) inhibits palmitate production and lipogenesis, and 2) shows evidence of clinical activity in KRAS mutant NSCLC patients. Additional biomarker and clinical analyses are ongoing in NSCLC and other tumor types. Citation Format: Marie O’Farrell, Tim Heuer, Katharine Grimmer, Richard Crowley, Joanna Waszczuk, Marina Fridlib, Richard Ventura, Claudia Rubio, Julie Lai, Doug Buckley, William McCulloch, George Kemble. FASN inhibitor TVB-2640 shows pharmacodynamic effect and evidence of clinical activity in KRAS-mutant NSCLC patients in a phase I study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-214.

Preclinical evaluation of novel fatty acid synthase inhibitors in primary colorectal cancer cells and a patient-derived xenograft model of colorectal cancer

Fatty Acid Synthase (FASN), a key enzyme of de novo lipogenesis, is upregulated in many cancers including colorectal cancer (CRC); increased FASN expression is associated with poor prognosis. Potent FASN inhibitors (TVBs) developed by 3-V Biosciences demonstrate anti-tumor activity in vitro and in vivo and a favorable tolerability profile in a Phase I clinical trial. However, CRC characteristics associated with responsiveness to FASN inhibition are not fully understood. We evaluated the effect of TVB-3664 on tumor growth in nine CRC patient-derived xenografts (PDXs) and investigated molecular and metabolic changes associated with CRC responsiveness to FASN inhibition. CRC cells and PDXs showed a wide range of sensitivity to FASN inhibition. TVB-3664 treatment showed significant response (reduced tumor volume) in 30% of cases. Anti-tumor effect of TVB-3664 was associated with a significant decrease in a pool of adenine nucleotides and alterations in lipid composition including a significant reduction in fatty acids and phospholipids and an increase in lactosylceramide and sphingomyelin in PDXs sensitive to FASN inhibition. Moreover, Akt, Erk1/2 and AMPK were major oncogenic pathways altered by TVBs. In summary, we demonstrated that novel TVB inhibitors show anti-tumor activity in CRC and this activity is associated with a decrease in activation of Akt and Erk1/2 oncogenic pathways and significant alteration of lipid composition of tumors. Further understanding of genetic and metabolic characteristics of tumors susceptible to FASN inhibition may enable patient selection and personalized medicine approaches in CRC.

Abstract A75: Translational studies of a first-in-class FASN Inhibitor, TVB-2640, linking preclinical studies to clinical laboratory observations in solid tumor patients

Dysregulated expression of FASN is a central mediator of neoplastic lipogenesis. FASN catalyzes the production of palmitate, the building block of long chain fatty acids, providing a mechanism to convert glucose and other carbon sources into lipids needed for production of phospholipids, formation of lipid rafts and the subsequent assembly of receptor tyrosine kinases and other signaling molecules in microdomains juxtaposed to the membrane. Inhibition of FASN in tumor cells leads to disruption of PI3K/Akt/mTOR and other important pathways leading to apoptosis. Targeting this critical nexus between lipogenesis and cellular survival/proliferation signaling pathways makes inhibition of FASN a novel therapeutic approach with a strong biological rationale and 3-V Biosciences has initiated clinical studies with its first-in-class FASN inhibitor. 3-V Bio has developed a series of orally available, reversible, potent, and selective FASN inhibitors that remodel tumor cell membranes, disrupt pAkt and Wnt/beta-catenin pathways, and reprogram gene expression. These effects lead to apoptosis of tumor cells in culture, inhibition of anchorage-independent cell growth under lipid-rich conditions and inhibition of in vivo xenograft tumor growth in mice and rats. We are currently using transcriptomic and metabolomic analyses, including lipidmomic profiling, to explore the fundamental mechanisms resulting in sensitivity to the FASN inhibitor, TVB-3166. These same platforms are used to examine the changes in response to FASN inhibition in tumor and surrogate tissues of xenograft models as well as surrogate tissues of cancer patients. A panel of tumor and normal cell lines was assembled and lipids were assessed prior to and 72 hours after treatment with TVB-3166. A reduction of saturated fatty acids was observed in all cell lines indicative of FASN inhibition. A similar extent of reduction was observed in media in the presence of 1% or 10% serum; these results demonstrated that the tumor cells did not compensate for FASN inhibition by increasing uptake of saturated fatty acids from the extracellular milieu. An inverse correlation was observed between the pretreatment quantities of saturated fatty acids and the induction of apoptosis resulting from FASN inhibition with TVB-3166; low levels of saturated fatty acids in cell lines such as COLO-205, correlated with a high level of cell killing. Normal, untransformed cells had significantly higher levels of pretreatment saturated fatty acids and a commensurate insensitivity to TVB-3166. These in vitro observations were extended to a COLO-205 rat xenograft model. Significant tumor growth inhibition was measured in these animals in response to FASN inhibition by TVB-3166. Tumor tissues were isolated and a reduction in pAkt and c-Myc expression were observed, the latter consistent with a reduction of Wnt/beta-catenin driven transcription. Further metabolomic analyses of surrogate and tumor tissues from this model continue. The power of the metabolomic/lipidomic and transcriptomic analyses to evaluate drug responsiveness was applied to surrogate tissues of a small number of cancer patients treated with the FASN inhibitor, TVB-2640. Following 8 days of oral dosing, a preliminary evaluation of aqueous metabolites and lipids in patients9 sera demonstrated changes consistent with the mechanism of FASN inhibition. Larger numbers of patients are being evaluated in order better characterize the extent of these changes. Preclinical evaluations of the molecular transformations caused by 3-V Bio9s FASN inhibitors are being translated into clinical assessments that may ultimately lead to a more complete understanding of the impact of FASN inhibition in cancer patients and potentially the identification of tumor types that are likely to respond to treatment. Citation Format: Doug Buckley, Tim Heuer, Marie O9Farrell, Bill McCulloch, George Kemble. Translational studies of a first-in-class FASN Inhibitor, TVB-2640, linking preclinical studies to clinical laboratory observations in solid tumor patients. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A75.

Abstract 4743: Preclinical studies characterize tumor type sensitivity to FASN inhibition and the mechanism and efficacy of novel drug combinations with TVB-2640

Tumor cells have an increased dependence on FASN-synthesized palmitate compared to non-tumor cells, which obtain many of their required lipids from the extracellular milieu. Palmitate and palmitate-derived lipids comprise diverse cellular components and function in processes required for tumor cell proliferation and survival. Previously we showed that FASN inhibition results in tumor cell apoptosis in vitro and xenograft tumor growth inhibition in vivo. Our studies demonstrated that diverse tumor types exhibit sensitivity to FASN inhibition and characterized mechanisms of action that associate with the antitumor activity of highly selective small molecule FASN inhibitors. In vitro studies with diverse tumor cell types elucidated a mechanism of action that includes plasma membrane remodeling, signal transduction pathway inhibition, and gene expression reprogramming. TVB-2640, TVB-3166, and TVB-3664 belong to a series of orally available, reversible, potent, and selective FASN inhibitors discovered and developed by 3-V Biosciences. Analysis of gene expression data from tumor cell lines and human tumors, both primary and patient-derived xenografts, has allowed for the classification of FASN sensitivity by tumor type, histology, and molecular genetic markers. Discoveries from these analyses are being characterized further using in vitro and in vivo studies. Combined inhibition of FASN and microtubule function with taxane treatment, e.g. paclitaxel, results in synergistic inhibition of tumor growth. Indeed, in Phase I clinical investigation, TVB-2640 combined with paclitaxel has shown promising early signs of clinical activity. Previous in vitro studies revealed that FASN inhibition causes changes in beta-tubulin expression and disrupts the organization of cellular microtubule structures in varied tumor cell types such as CALU-6 non-small-cell lung and 22Rv1 prostate tumor cell lines. Extending our investigation of the mechanism of FASN/taxane synergy, we now show that FASN inhibition prevents beta-tubulin palmitoylation. This likely plays a significant role in the observed effects on beta-tubulin expression and microtubule architecture. As disruption of protein palmitoylation is believed to contribute significantly to the anti-tumor activity of FASN inhibition in general, we expanded the analysis of protein palmitoylation following inhibition of FASN with TVB-3166 and TVB-3664 to include key oncogenic drivers of cell growth, proliferation, and survival such as K-Ras and EGFR. Additionally, the efficacy of FASN inhibition in combination with additional, non-taxane approved cancer therapies, including immunomodulatory agents and bevacizumab, is being investigated. Citation Format: Timothy S. Heuer, Richard Ventura, Julie Lai, Joanna Waszczuk, Claudia Rubio, Glenn Hammonds, Marie O’ Farrell, Douglas Buckley, George Kemble. Preclinical studies characterize tumor type sensitivity to FASN inhibition and the mechanism and efficacy of novel drug combinations with TVB-2640. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4743.

Abstract 1010: Evaluation of small-molecule FASN inhibitors in preclinical models of colorectal cancer

Fatty Acid Synthase (FASN), a key enzyme of de novo lipid synthesis, is upregulated in many cancers including colorectal cancer (CRC); increased FASN activity is associated with decreased survival and increased disease recurrence. Recently, a first-in-class, oral FASN inhibitor (TVB-2640) entered a Phase I clinical trial (3V2640-CLIN-002) in solid tumor patients demonstrating a favorable tolerability profile with no significant adverse events; however, tumor characteristics that would indicate responsiveness to FASN inhibition are not fully understood. The purpose of our study was: (i) to determine the effect of novel, selective and reversible FASN inhibitors on proliferation of primary CRC cell cultures, established CRC cell lines, and CRC patient-derived xenografts (PDXs); and (ii) to identify potential biomarkers associated with CRC responsiveness to FASN inhibition. METHODS. The effect of TVB-3166, TVB-3664, and TVB-3693 (all developed by 3-V Biosciences) on the proliferation of primary cells (established from 1 st generation PDX tumors) and CRC cell lines was assessed by cell count; apoptosis was assessed by Cell Death ELISA. In addition, tumor growth was assessed in PDX models established in NOD SCID gamma mice using freshly resected CRC specimens (either primary CRC or metastasis) from our patient population. Once the xenografts grew to ∼100 mm 3 , mice were randomized into two groups (n = 5) to receive either vehicle or TVB-3664 (3mg/kg) by gavage daily. Tumor volume and animal weights were measured weekly. Western blot analysis, immunohistochemistry and immunofluorescent staining were used to identify FASN-mediated changes in β-catenin, Akt and AMPK pathways. RESULTS. TVB compounds tested showed similar efficacy in primary and established CRC cells with a wide range of sensitivity to FASN inhibition. The 5 cell lines that were most responsive to FASN inhibition demonstrated a low basal level of pAMPK and pAkt as compared to the 5 least responsive cells. Moreover, we noted that increased FASN protein expression was also associated with increased sensitivity to FASN inhibition. Inhibition of proliferation by TVB compounds was associated with decreased expression of active β-catenin, c-MYC, pAkt, and survivin, while an increase in apoptosis was noted by induction of PARP cleavage. Consistent with our in vitro studies, TVB-3664 treatment significantly reduced tumor volume in vivo with no weight changes or toxicity observed. CONCLUSIONS. Our studies show that the novel FASN inhibitors, as a single agent, significantly inhibit CRC growth both in vitro and in vivo. Importantly, our results suggest that basal activation of AMPK and Akt may be predictive of responsiveness to FASN inhibition and may function as potential biomarkers to allow a more personalized treatment approach. Citation Format: Yekaterina Y. Zaytseva, Piotr G. Rychahou, Tianyan Gao, Eun Y. Lee, Heidi L. Weiss, Timothy S. Heuer, George Kemble, B. Mark Evers. Evaluation of small-molecule FASN inhibitors in preclinical models of colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1010.

Abstract C175: FASN inhibition studies in preclinical tumor models identify biomarkers that align with in vitro and in vivo sensitivity to TVB-2640

Tumor cells have an increased dependence on FASN-synthesized palmitate compared to non-tumor cells, which obtain many of their required lipids from the extracellular milieu. Palmitate and palmitate-derived lipids comprise diverse cellular components and function in processes required for tumor cell proliferation and survival. Previously we showed that FASN inhibition results in tumor cell apoptosis in vitro and xenograft tumor growth inhibition in vivo. Our studies demonstrated that diverse tumor types exhibit sensitivity to FASN inhibition and characterized mechanisms of action that associate with the antitumor activity of highly selective small molecule FASN inhibitors. In vitro studies with diverse tumor cell types elucidated a mechanism of action that includes plasma membrane remodeling, signal transduction pathway inhibition, and gene expression reprogramming. TVB-2640 and TVB-3166 belong to a series of orally available, reversible, potent, and selective FASN inhibitors discovered and developed by 3-V Biosciences. Tumor xenograft studies were conducted in rats and mice to examine the in vitro and in vivo relationship of tumor cell sensitivity to FASN inhibition. Pharmacodynamic analyses of tumor and serum samples from these studies characterized the mechanism of action and biomarkers of sensitivity to in vivo FASN inhibition. Once daily oral dosing of TVB-2640 or TVB-3166 caused inhibition of xenograft tumor growth for varied tumor models that included COLO-205 and HCT-116 colon adenocarcinoma cell lines. In vivo sensitivity to FASN inhibition was in agreement with in vitro data. Analysis of lipid, metabolite, protein, and RNA expression in tumor, blood or serum samples showed drug-induced modulation that was consistent with independent in vitro or in vivo studies. Lipid and metabolite changes included decreased palmitate and palmitate-associated lipids as well as increased expression of acylcarnitine species. Decreased expression of pAkt (S473), β-catenin, pβ-catenin (S675), and Myc proteins were found to associate quantitatively with xenograft tumor growth inhibition. Additionally, mRNA expression was modulated in a manner that revealed coordinated changes in the mRNAs from fatty acid, metabolism, cell survival, and cell growth-associated pathways. Expression changes in lipids, metabolites, proteins, and RNA species are leading to the development of a biomarker panel that describes FASN inhibitor target engagement and tumor sensitivity in both in vitro and in vivo studies. These mechanism-based marker panels will be evaluated in current and upcoming clinical studies of TVB-2640. Citation Format: Timothy S. Heuer, Richard Ventura, Kasia Mordec, Julie Lai, Joanna Waszczuk, Claudia Rubio, Marie O9 Farrell, Douglas Buckley, George Kemble. FASN inhibition studies in preclinical tumor models identify biomarkers that align with in vitro and in vivo sensitivity to TVB-2640. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C175.

Abstract B13: Biomarker Analyses from Dose Escalation Phase of FASN Inhibitor TVB-2640 Phase 1 Study Shows Target Engagement in Solid Tumor Patients

TVB-2640 is a potent, reversible and selective fatty acid synthase (FASN) inhibitor currently in Phase 1 in solid tumor patients (study 3V2640-CLIN-002). This study includes biomarker assays to assess pharmacodynamic activity and refine strategies for patient selection. This report focuses on biomarker results from the dose escalation phase to assess pharmacodynamic activity for this first in class agent. FASN is a central mediator of neoplastic lipogenesis, and uniquely catalyzes the production of palmitate, the building block of long chain fatty acids. Unlike most normal cells, tumor cells frequently have high FASN expression, which correlates with poor prognosis in several tumor types. Tumor cells rely on de novo lipogenesis to produce phospholipids, lipid second messengers, membranes and lipid rafts needed for oncogenic signaling, survival and proliferation, via many pathways including activated RTKs and hormone receptors. In Phase 1 study 3V2640-CLIN-002 (NCT02223247), 31 patients were enrolled on TVB-2640 monotherapy (oral, once daily) and 13 patients were enrolled in combination with weekly paclitaxel. Plasma TVB-2640 drug levels increased with dose with a half-life of approximately 15 hr. Several complementary biomarker approaches were pursued to investigate FASN pathway inhibition in tumor and surrogate tissues. (i) Serum was assessed by mass spectrometry-based global metabolomic profiling to screen for changes following TVB-2640 administration. Increased levels of malonyl carnitine were observed after 8 days of TVB-2640 treatment in 9/10 patients tested. The degree of change of malonyl carnitine increased with greater exposure to the drug. Decreased levels of tripalmitin were also observed in 9/10 patients. These changes are consistent with the expected consequences of FASN inhibition. Serum metabolite profiles from rats administered a FASN inhibitor for 5 to 7 days showed similar trends. These data provide evidence of FASN inhibition by TVB-2640, a first in class agent, in the clinic. (ii) Fresh tumor biopsies were analyzed by immunohistochemistry (IHC) for markers relevant to FASN engagement. In all 4 patients to date with evaluable biopsies, pAKT S473 decreased by 30-50% after 1 cycle of TVB-2640 treatment relative to predose. Total AKT did not change in either of the 2 patients tested. pAKT inhibition has also been observed in preclinical rat xenografts treated with TVB-2640. (iii) Gene expression analysis by RNA Seq has been initiated in pre and post dose macro-dissected tumor biopsies in a limited number of patients, and in whole blood for up to 13 patients. Preliminary bioinformatics analyses show modulation of several lipid metabolism pathways consistent with preclinical studies. In conclusion, FASN pathway inhibition has been demonstrated at well tolerated dose levels of TVB-2640 in both tumor and serum from cancer patients. Increased serum malonyl carnitine and decreased levels of palmitate derivatives provide easily accessible biomarkers and show target inhibition. Early tumor biopsy analysis provides evidence of FASN inhibition in the target tissue by IHC and gene expression analysis. These and additional biomarker studies will be conducted in the expansion phase of this study and help guide clinical development of this first in class FASN inhibitor. Citation Format: Marie O9Farrell, Richard Crowley, Timothy Heuer, Marina Fridlib, Doug Buckley, William McCulloch, George Kemble. Biomarker Analyses from Dose Escalation Phase of FASN Inhibitor TVB-2640 Phase 1 Study Shows Target Engagement in Solid Tumor Patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B13.